Anti cd45 percp cyanine5

Mouse livers were perfused with HBSS via the portal vein and followed by 0.04% collagenase IV (C5138, Sigma, MO, USA). Perfused livers were dissected and teased upon 70-μm cell strainers, then suspended in 40 mL DMEM supplemented with 10% FBS. Non-parenchymal cells (NPCs) were separated from hepatocytes by centrifugation at 50 × g for 2 minutes three times. NPCs were plated in cell culture dishes in DMEM supplemented with 10% FBS, 10 mM HEPES, 2 mM GlutaMax, 100 U/mL penicillin, and 100 mg/mL streptomycin for 15 minutes at 37°C, then the non-adherent cells were removed. The CD45⁺F4/80⁺ macrophages adherent cells were used for Flow cytometry. Antibodies used were anti-CD45-PerCP/Cyanine5.5 (103131, Biolegend, CA, USA), anti-CD11b-BV510 (101263, Biolegend, CA, USA), anti-F4/80-APC (123116, Biolegend, CA, USA), anti-CD206-PE (141706, Biolegend, CA, USA) and anti-CD86-BV605 (105037, Biolegend, CA, USA). Cells stained with corresponding isotype control antibodies were used as controls. Data were analyzed using CytExpert software (Beckman, CA, USA).